The overall direction of the Molecular Mechanisms of Tumor Promotion Section is to understand the regulation of the signalling pathways downstream from the lipophilic second messenger diacylglycerol, to elucidate the basis for heterogeneity of response to different ligands which function through this pathway, and to exploit this understanding for developing novel ligands with unique behaviour that function through this pathway. A complementary direction is to understand the regulation and structure activity relations for the vanilloid receptor. The vanilloid receptor is a downstream target of the diacylglycerol signalling pathway, shares partial homology in its ligands to this pathway, and shares with the diacylglycerol signalling pathway an important role in inflammation. Both directions impact both our understanding of biological regulation and the potential development of therapeutic agents for cancer chemotherapy or for supportive care for cancer patients. Exploiting strong collaborations with groups in computational chemistry and synthetic chemistry, we continue to improve our understanding of the structural basis for ligand - protein kinase C interactions. Reflecting the important role revealed for the side chains of ligands in controlling the membrane localization of some PKC isoforms, we are currently focusing on their modification. We have optimized the linker length for dimers to obtain compounds with nM affinity. We are evaluating derivatives in which the side chains, unlike those in typical phorbol esters, are rigid and therefore less able to interact with the membrane lipids. We are exploring derivatives embodying different hydrophilic moieties in the otherwise hydrophobic side chain. We are exploring the roles of the postulated elements of the pharmacophore in the ligand interaction and trying to add additional pharmacophoric elements. Natural products provide a complementary approach to medicinal chemistry for the identification of novel structures with novel biological properties. Ingenol 3-angelate, derived from E. peplus, has been identified as a potential chemotherapeutic agent for non-melanoma skin cancer. We find that ingenol 3-angelate shows unique activity on PKC, stabilizing its association with membranes only weakly compared to PMA. In competition with PMA, ingenol 3-angelate partially reverses the stabilization exerted by PMA, thereby functioning as a partial agonist. Ingenol 3-angelate is the first example of a potent ligand for PKC which acts in this fashion. Consistent with the novel pharmacological behavior of ingenol 3-angelate, we find that ingenol 3-angelate induces the cytokine IL-6 to a greater extent than does phorbol 12-myristate 13-acetate, a typical phorbol ester. Protein kinase C regulation is marked by multiple levels of control. Using green fluorescent labeled protein kinase C, we are able to visualize subcellular localization as a function of ligand. By varying the lipophilicity within a homologous series of compounds, we can show that lipophilicity is one of the determinants of subcellular localization. Using fluorescent phorbol esters, we can now compare the kinetics of uptake and localization of ligand with that for the translocation of PKC isoforms. Tyrosine phosphorylation of protein kinase C delta is a second important regulatory factor, and different sites of phosphorylation control different biological responses mediated by the enzyme. In glioma cells, we show that different tyrosine residues are involved in the apoptosis response to etoposide and to Sindbis virus and that PKC delta has opposite effects on apoptosis induced by these two agents. Vanilloid receptors are important mediators of C-fiber mediated pain and represent a promising therapeutic target for treatment of pain. As part of a strong collaborative effort, we have developed ligands with high potency and novel properties. Of particular interest, we have identified compounds that function as potent, partial agonists. We show that their degree of partial agonism (and their reciprocal partial antagonism) depends on the presence of co-stimulators. Under appropriate conditions, we can thus convert a compound which has almost no agonist activity into one with almost complete agonist activity. An underlying problem for desensitization of vanilloid receptors is that often one would only wish to desensitize the receptors at a site of inflammation. The ability of co-stimulators at a site of inflammation to convert a compound into an active agent only at that site might represent a solution to this problem. In other studies, we have characterized a novel antagonist that only functions on the plasma membrane located pool of the vanilloid receptor, while having negligible affinity for that receptor involved in release of calcium from internal pools. These studies prove the feasibility of dissecting subpopulations of vanilloid receptors and probing their physiological functions.